Abstract
Objective
To investigate the intracellular localization and binding of duck invariant chain (Ii) to major histocompatibility complex (MHC) class Iα or MHC class IIβ molecules.
Methods
First, the genes of duck Ii, MHC Iα, and MHC IIβ were amplified by PCR, cloned into expression plasmids, and then sequenced to compare homology between ducks and selected agricultural species. Second, Rosetta or 293T cells were transfected with plasmids encoding Ii, MHC Iα, and MHC IIβ either alone or in combination to interrogate binding following expression. The fluorescent reporter molecules used to tag/image transfected cells and expressed protein were identified by western blot. Additionally, the binding interaction of the expressed products was observed by affinity purification using pull-down assay and coimmunoprecipitation. Finally, intracellular colocalization was observed by laser confocal microscopy.
Results
The results of western blots demonstrated that all constructed plasmids successfully expressed proteins. Pull down and coimmunoprecipitation revealed that Ii could bind Iα or IIβ, forming Ii-Iα or Ii-IIβ complexes, and they dissociated into single molecules after SDS treatment. In 293T cells, Ii interacts with MHC Iα or MHC IIβ chains and colocalizes intracellularly. Confocal fluorescence microscopy assay indicated that MHC Iα and IIβ were located in the endoplasmic reticulum and endosome.
Conclusions
Both MHC and Ii proteins exhibit direct interactions and can colocalize in the endoplasmic reticulum and endosomes.
Clinical Relevance
This study investigates the relationship between duck Ii and MHC proteins, offering a theoretical foundation for exploring the role of Ii in immune responses and advancing the development of Ii-vectored avian vaccines.
The major histocompatibility complex (MHC) is mainly comprised of MHC class I and II molecules, which are responsible for presenting endogenous and exogenous antigenic peptides to T cells, respectively.1,2 Thus, it plays a key role in initiating specific immunity. Major histocompatibility complex class I and II molecules are heterodimers composed of α and β chains. Among them, the α1α2 domain of MHC Iα chain and β1 domain of MHC IIβ chain contain a highly polymorphic peptide-binding region, which serves as the primary domain for antigen binding.3,4 Major histocompatibility complex loci are among the most polymorphic regions in the genomes of vertebrates.5 Polymorphisms within MHC genes result in structural variations of MHC molecules that modulate their binding affinities to diverse antigenic peptides.6 This variability may influence T-cell receptor recognition of antigen-MHC complexes, thereby altering the intensity and specificity of immune responses. Notably, specific allelic variants exhibit preferential binding to pathogen-derived epitopes, which may confer enhanced resistance against corresponding pathogens in genetically predisposed individuals.7–9 Avian MHC molecules are characterized by their simplicity and diversity, making them a prominent nonmammalian vertebrate model for studying MHC structure and genomic organization.10–12 The diversity of the avian MHC is linked to disease resistance via allelic diversity and functional gene expression changes. Studies in migratory songbirds show that higher MHC class I allelic diversity reduces malaria parasite infection, with specific alleles, like B4B, associated with protective immune responses. In house finches, rapid resistance to Mycoplasma infection involves MHC-linked transcriptional changes that enhance survival.13,14 The MHC structure of ducks is similar to that of chickens, primarily consisting of MHC class I and II molecules. Polymorphic sites are mainly located in the α1α2 domain of MHC Iα and the β1 domain of MHC IIβ.15,16 Currently, investigations into duck MHC II molecules remain relatively scarce, with the majority of research efforts concentrated on the polymorphism of MHC I molecules and their relationship with disease resistance.17 It has been confirmed that duck MHC I molecules can present antigenic peptides derived from highly pathogenic influenza A and duck Tembusu virus, thus activating immune responses.18–20 Despite these existing studies on duck MHC I, the comprehension of its polymorphism and functional classification across various duck breeds is still insufficient. This gap in knowledge poses a significant bottleneck in advancing the understanding of cytotoxic T-cell–mediated immunity in ducks.
The invariant chain (Ii) serves as a chaperone molecule for MHC II molecules. The class II–associated Ii peptide (CLIP) region at the N-terminus of Ii is embedded into the peptide-binding groove of MHC IIαβ heterodimers, forming an Ii3:(αβ)3 complex. This complex is subsequently transported to endosomes (endos), where the CLIP is replaced by antigenic peptides, ultimately facilitating the processing and presentation of exogenous antigens.21 Additionally, the Ii enhances the assembly and transport of MHC II molecules.22 The Ii can also assist MHC I in the presentation of exogenous antigens to CD8+ T cells and the crosspresentation of antigens by MHC I molecules.23–25 Due to the immunomodulatory role of the Ii, vaccine strategies using Ii as a vector have been studied. Two approaches—constructing Ii-antigen chimeras by replacing its CLIP region with antigens or developing adenoviral/lentiviral vector–based vaccines—enhance cellular (CD4+/CD8+ T cell) and humoral immune responses. For example, Ii-fused tumor/Zika virus antigens boost protective immunity, whereas Ii-adjuvanted viral vectors enhance malaria-specific antibody titers and human T-cell activation. These strategies leverage Ii’s ability to improve antigen presentation and immune cell targeting.26–31 In addition, Ii possesses other functions, such as playing a crucial role in sorting MHC class I and II molecules, along with other related molecules, such as fragment crystallizable receptors, into specific endocytic pathways.32 Furthermore, Ii is involved in the regulation of B-cell development and maturation, and it can act as a receptor for cytokines and viruses.33–35
Although the structure, colocalization, and binding of Ii to MHC II molecules and its role in immunology have been well characterized in various species, such as chickens, humans, mice, and fish, it remains unclear whether there is a colocalization and binding relationship between Ii and MHC in waterfowl. The binding relationship between recombinantly expressed duck MHC and Ii was interrogated by affinity chromatography using pull-down and coimmunoprecipitation (co-IP) assays. Additionally, the distribution of these molecules in organelles was observed through eukaryotic expression, allowing for a better understanding of the intracellular localization characteristics of MHC molecules and Ii. This study laid the foundation for further research on the functions of waterfowl MHC and Ii in antigen processing and presentation.
Methods
Cell lines and cell culture
Human embryonic kidney 293T cell lines were obtained from the ATCC. The 293T cells were cultured in Dulbecco’s modified Eagle medium (Hyclone) supplemented with 10% FBS (Hyclone) and were cultured at 37 °C in a humidified atmosphere of 5% CO2.
Recombinant vector construction for duck MHC and Ii genes, PCR, and cross-species analysis
To investigate the structural characteristics of waterfowl MHC and Ii, the gene fragments of duck Ii, MHC Iα, and MHC IIβ were cloned and inserted into prokaryotic (pGEX-4T-1 or pET-32a) and eukaryotic (pmCherry-N1 or pEGFP-N1) expression plasmids, respectively. The target genes were constructed into prokaryotic and eukaryotic vectors by double enzyme digestion, and then the recombinant plasmids were transferred into Escherichia coli (DH5α) to construct recombinant bacteria. Duck Ii, MHC Iα, and MHC IIβ genes were amplified by PCR with self-designed primers according to the sequences in GenBank (HQ909102A, AB115244, and DQ490138) from the cDNA derived from the spleen tissue of duck (Anas platyrhynchos). The PCR products that were purified by agarose gel and the empty vectors were digested by double enzyme digestion. Then, all of them were linked by Ligation High Ver.2 (TOYOBO) to construct recombinant plasmids, which were identified by PCR and sequencing (Huada). The sequences of the primers, the restriction endonucleases (Takara), and the recombinant plasmids are shown in Supplementary Table S1. The methods for constructing the recombinant plasmids were in accordance with our previous report.36 Furthermore, MEGA software (version 11; https://www.megasoftware.net/) was used to analyze the homology among the ducks, chickens, pigs, and cattle MHCIα, MHCIIβ, and Ii amino acid sequences. Amino acid sequences of target genes were retrieved from the National Center for Biotechnology Information (NCBI) using accession numbers. Multiple sequence alignment was performed with MEGA11 using ClustalW parameters optimized for phylogenetic analysis. Amino acid homology of the 3 genes across species was calculated using MegAlign (DNAStar). The accession numbers for the amino acid sequences retrieved from the NCBI are as follows: Ii of duck (AAX89536), chicken (AAT36345), pig (BAI47604), and cattle (BAA12156); MHC Iα of duck (AAQ62600), chicken (AGW80460), pig (BAK52508), and cattle (AHI12882); and additional MHC IIβ for duck (ABF47549), chicken (ADR71219), pig (ACA21792), and cattle (AAB36532).
Expression and purification of recombinant proteins in E coli using glutathione S-transferase and polyhistidine tags
The recombinant plasmids pET-32a-Ia, pET-32a-IIβ, and pGEX-4T-1-Ii, along with corresponding empty vectors (pET-32a and pGEX-4T-1), were individually transformed into competent-cell E coli Rosetta(DE3) (Beyotime) to generate 5 recombinant bacteria strains. The strains confirmed by PCR and sequencing were further subjected to protein expression. Each recombinant bacterial strain was inoculated into Luria-Bertani (LB) solid culture medium (Beyotime) and incubated at 37 °C overnight. A single positive colony was selected and inoculated into 4 mL LB medium containing 100 μg/mL ampicillin (Beyotime) for shaking culture at 37 °C and 180 revolutions/min overnight. Subsequently, 30 μL of the overnight culture was transferred to 300 mL fresh LB medium (100 μg/mL ampicillin) and grown under identical conditions until the optical density at 600 nm reached 0.6. Protein expression was induced by adding isopropyl-β-D-thiogalactoside (Beyotime) to a final concentration of 200 μg/mL, followed by incubation at 16 °C and 180 revolutions/min for 16 hours. Finally, the recombinant bacteria after induction were harvested by centrifugation (10,000 X g, 4 °C, 20 minutes). The bacterial pellets were washed with ice-cold PBS and lysed via ultrasonication (180 W, 5-second pulse/10-second interval, 60 cycles). Lysates were centrifuged (12,000 X g, 15 minutes, 4 °C) to pellet inclusion bodies. The inclusion bodies were denatured by dissolution with 8 mol/L urea (Smart-Lifesciences) and dialysis in PBS for 48 hours, replacing the dialysis solution every 4 hours to enable protein renaturation. The protein glutathione S-transferase (GST)/Ii expressed in pGEX-4T-1-Ii was purified by a glutathione agarose 4B column (Smart-Lifesciences). The purified GST/Ii protein was obtained using 10 mmol/L reduced glutathione elute resin. The proteins polyhistidine (His)/Iα and His/IIβ expressed by pET-32a-Iα and pET-32a-IIβ were purified using a Ni column (Smart-Lifesciences). The protein was eluted by 10, 20, 40, 60, 80, 100, and 500 mmol/L imidazole (Sigma-Aldrich), respectively. Finally, the eluted protein was stored at −20 °C for further use. Before and after protein purification, 5 μL of each protein sample (renatured inclusion body protein before purification and purified protein after purification) was mixed with an equal volume of 5X SDS-PAGE protein loading buffer (Beyotime), boiled for 10 minutes, and analyzed by SDS-PAGE.
Pull-down assay demonstrating binding between Ii and both MHC Iα and MHC IIβ
For His-tag pull down, the proteins His/Iα, His/IIβ, and His were bound to Ni Sepharose (GE Healthcare) for 1 hour at 4 °C. Then, the GST/Ii or GST proteins were added and incubated for 3 hours at 4 °C. After washing with cold PBS, proteins were released with elution buffer (20 mmol/L sodium phosphate containing 100 mmol/L imidazole, pH 7.4), and then we collected the proteins, mixed them with 5X loading buffer, and denatured them by boiling for 10 minutes. We separated the samples by native PAGE and SDS-PAGE.
Then, the samples were transferred to 0.22-μm polyvinylidene fluoride membranes (Millipore) using a transfer buffer at 100 V for 2 hours. We incubated the membranes with Tris-buffered saline containing Tween 20 (TBST) and 5% bovine serum albumin for 1 hour at room temperature and then incubated the membranes with primary anti-His or GST monoclonal antibody (M20001 or M20007; Abmart) at 4 °C overnight. Then, we washed them 3 times with TBST, and then followed by a goat polyclonal horseradish peroxidase–labeled secondary antibody against mouse immunoglobulin G (Heavy + Light chain; ZB-2305; ZSGB) for 1 hour at room temperature. After the membranes were washed 3 times with TBST, the protein was detected by enhanced chemiluminescence solution (Beyotime) and visualized using the chemiluminescence detection system (Bio-Rad).
Coimmunoprecipitation assay—Before transfection, 293T cells were seeded in 6-cm dishes. The plasmids pmCherry-N1-Iα, pmCherry-N1-IIβ, and pmCherry-N1 were individually cotransfected with pEGFP-N1-Ii into 293T cells; similarly, pEGFP-N1-Iα and pEGFP-N1-IIβ were separately cotransfected with pmCherry-N1 into 293T cells. After coincubation for 36 hours, cells from each well were individually harvested, washed with ice-cold PBS, and lysed in buffer (Beyotime) containing 1mM phenylmethylsulfonyl fluoride, 1mM dithiothreitol, and a protease inhibitor cocktail (Biolinkedin). The lysates were clarified by centrifugation at 12,000 X g for 10 minutes at 4 °C, and the supernatant was split into 2 fractions: 1 aliquot served as the input control, and the remainder was incubated with anti–green fluorescent protein (GFP) antibody-conjugated protein A/G magnetic beads (Biolinkedin) at 4 °C for 3 hours under gentle rotation. Following 5 sequential washes with PBS containing protease inhibitors, bead-bound complexes were eluted by boiling in 5X SDS loading buffer at 95 °C for 10 minutes. Protein interactions between monomeric Cherry red fluorescent protein (mCherry)-tagged MHCIα/IIβ and GFP-tagged Ii were resolved by SDS-PAGE and western blot with mCherry-Tag (clone 4C16) and GFP-Tag (clone 7G9) mouse monoclonal antibodies (Abmart).
Confocal fluorescence microscopy
The subcellular localization of MHC Iα, MHC IIβ, and Ii within organelles (endoplasmic reticulum [ER] and endo) was validated using confocal microscopy. Before transfection, 293T cells were cultured in Dulbecco’s modified Eagle medium supplemented with 10% FBS and 1% penicillin-streptomycin (Gibco) at 37 °C, 5% CO2. Cells were seeded in 24-well plates at a density of 1 X 105/well overnight. The recombinant plasmids with mCherry-fused pmCherry-N1-Iα, pmCherry-N1-IIβ, and pmCherry-N1-Ii were cotransfected with GFP-fused organelle markers plasmids pEGFP-N1-ER (WZ Biosciences) or pEGFP-N1-endo (WZ Biosciences) into 293T cells. After 36 hours, the cells were fixed with 4% paraformaldehyde for 30 minutes at 4 °C. The cells were washed with PBS 3 times. The fixed cells were observed under an LSM880 confocal laser-scanning microscope (Carl Zeiss AG) using excitation wavelengths of 488 and 587 nm to detect the emission wavelengths of 509 nm (green, likely from GFP) and 610 nm (red, likely from mCherry), respectively. If mCherry-tagged MHCIα, MHCIIβ, or Ii colocalize with GFP/endo or GFP/ER in cellular compartments, they will exhibit orange fluorescence due to red-green colocalization; otherwise, they retain their original red or green fluorescence, respectively. The cell culture and transfection methods are according to our previous report.36
Results
Analysis of structural characteristics of duck MHC Iα, MHC IIβ, and Ii
Duck Ii, MHC Iα, and MHC IIβ gene fragments were obtained by PCR amplification. The target fragments were 669, 1,074, and 780 bp, respectively. After PCR and sequencing, it was confirmed that the constructed recombinant plasmids were correct. The amino acid sequences and protein domains of duck Ii, MHC Iα, and MHC IIβ were compared with those of other species (chickens, pigs, and cattle) using the MEGA11 software. The amino acid sequences and their corresponding structural domains of MHC Iα, MHC IIβ, and Ii of ducks, chickens, pigs, and cattle were obtained from the NCBI, respectively. It was revealed that duck Ii molecules in all species contain transmembrane, cytoplasmic, CLIP, and trimer regions (Figure 1). Similarly, MHC Iα consists of transmembrane, α1, α2, and α3 regions. Major histocompatibility complex IIβ contains transmembrane, β1, and β2 regions. The comparison of amino acid sequences showed variations in homology between ducks and other species (chickens, pigs, and cattle). Through the calculations performed by DNAstar, it was revealed that the homologies of MHC Iα amino acids between ducks and the 3 species were 61.95%, 43.41%, and 43.05% for the respective species. Similarly, the homologies of MHC IIβ amino acids were 67.74%, 49.40%, and 52.51%, and the homologies of Ii amino acids were 67.28%, 51.91%, and 47.54% for the respective species. These findings demonstrate conserved protein structures and domain architectures across duck, chicken, pig, and cattle orthologs of the 3 genes, and their amino acid homologies exhibit species-specific divergence.
Amino acid comparison of different species invariant chain (Ii), major histocompatibility complex (MHC) Iα, and MHC IIβ. A—Aligned amino acid sequences of Ii between different species. The gray background indicates the class II–associated Ii peptide area. B—Aligned amino acid sequences of Iα between different species. The gray background indicates the α1α2 area. C—Aligned amino acid sequences of IIβ between different species. The gray background indicates the β1 area. National Center for Biotechnology Information (NCBI) accession numbers of Ii: duck, AAX89536; chicken, AAT36345; pig, BAI47604; and cattle, BAA12156. The NCBI accession numbers of MHCIα: duck, AAQ62600; chicken, AGW80460; pig, BAK52508; and cattle, AHI12882. The NCBI accession numbers of MHC IIβ: duck, ABF47549; chicken, ADR71219; pig, ACA21792; and cattle, AAB36532. *The same amino acid.
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.12.0394
The binding biological activity of duck MHC Iα, MHC IIβ, and Ii protein molecules is exhibited in both prokaryotic and eukaryotic expression systems
The binding activity of Ii to MHC was detected by pull down and co-IP following expression in Rosetta cells. Sodium dodecyl sulfate PAGE analysis of isopropyl-β-D-thiogalactoside–induced recombinant bacterial strains revealed distinct protein bands at 57.7, 46.7, and 50.8 kDa, consistent with the expected molecular weights of His/Iα, His/IIβ, and GST/Ii, respectively. However, several nonspecific bands were detected in the unpurified proteins, which affected the purity of the proteins (Figure 2). To further improve the purity of the proteins, affinity chromatography was employed for purification. Subsequent SDS-PAGE analysis demonstrated that the purified proteins exhibited significantly higher purity compared to the unpurified counterparts.
A—Identification of prokaryotic expression products and the complex of duck Ii-Iα and Ii-IIβ. Following isopropyl-β-D-thiogalactoside induction, the recombinant bacterial strains transformed with plasmids pET-32a-Iα, pET-32a-IIβ, and pGEX-4T-1-Ii expressed the corresponding polyhistidine (His)/Iα, His/IIβ, and GST/Ii proteins, respectively. Lanes 2, 3, and 4 show His/Iα, His/IIβ, and GST/Ii, respectively. B—The proteins His/Iα and His/IIβ purified via nickel affinity chromatography, along with GST/Ii purified through the glutathione agarose 4B column, were identified by SDS-PAGE analysis. Lanes 2, 3, and 4 show His/Iα, His/IIβ, and GST/Ii, respectively. GST = Glutathione S-transferase. M = Protein marker.
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.12.0394
To assess interactions between refolded MHC Iα, IIβ, and Ii, His-MHC Iα/IIβ or His was immobilized on Ni Sepharose, followed by the addition of GST-Ii or GST. Eluted proteins were analyzed by native/SDS-PAGE and western blot. In the native PAGE analysis without SDS treatment (Figure 3), lane 2 exhibited bands corresponding to GST/Ii-His/Iα (108.5 kDa) and His/Iα (57.7 kDa), suggesting the formation of a complex and potential binding interaction. Lane 3 showed bands for GST/Ii-His/IIβ (97.5 kDa) and His/IIβ (46.7 kDa), indicating a similar binding relationship. Polyhistidine (20.9 kDa) in lane 4 served as a control with a single detected band, confirming the reliability of the assay. Overall, these results provide evidence for the binding interactions between the studied proteins, laying a foundation for further understanding their functional roles in relevant biological processes.
Identification by native PAGE of the complex of duck Ii-Iα and Ii-IIβ in pull down. Lanes 2, 3, and 4 show His/Iα-GST/Ii, His/IIβ-GST/Ii, and His, respectively.
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.12.0394
Furthermore, the eluted protein complexes GST/Ii-His/Iα and GST/Ii-His/IIβ were separately mixed with an equal volume of 5X SDS-PAGE protein loading buffer, followed by analysis using SDS-PAGE under reducing conditions, which is in contrast to native PAGE performed in nonreducing conditions. After SDS treatment, the 2 complexes dissociated into single molecules: GST/Ii and His/Iα or GST/Ii and His/IIβ (Figure 4). The molecular weights of His/Iα, GST/Ii, and His/IIβ protein bands each corresponded to those of their respective positive controls. Finally, to verify the specificity of the protein, the single molecules were detected by western blots. Polyhistidine/Iα-GST/Ii and His/IIβ-GST/Ii complexes formed in the pull-down assay were dissociated into single molecules by SDS and specifically bound by anti-His and anti-GST antibodies as demonstrated by western immunoblot. These results indicated that the duck Ii is capable of physically binding to MHC Iα or MHC IIβ molecules.
A—Identification by SDS-PAGE and western blots of the dissociation of duck Ii from Iα and IIβ. Identification by SDS-PAGE of the dissociation of duck Ii from Iα and IIβ. Lanes 3 and 5 show protein complexes GST/Ii-His/Iα and GST/Ii-His/IIβ, respectively. Lanes 2, 4, and 6 show His/Iα, GST/Ii, and His/IIβ, respectively. B and C—Identification of Ii and Iα or IIβ in the complex by western blots. After the pull down, the eluted products were collected and detected subsequently by SDS-PAGE, transfer electrophoresis, and western blot with anti-His ([B] lane 2 shows His/Iα, lane 3 shows His/IIβ, and lane 4 shows His) or anti-GST ([C] lane 2 is GST/Ii, and lane 3 is GST).
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.12.0394
To further investigate the binding capability of duck Ii to MHC Iα or MHC IIβ in eukaryotic expression systems, we performed co-IP assay. Figure 5 presents the outcomes in this experiment. In the co-IP group, the GFP antibody detected GFP/Ii (51.1 kDa) and GFP (26.3 kDa) on beads, confirming their immobilization. The mCherry antibody specifically identified mCherry/Iα (62.8 kDa) and mCherry/IIβ (51.8 kDa) coimmobilized with GFP/Ii on the beads, whereas mCherry alone was not detected, suggesting specific interactions between GFP/Ii and both mCherry/Iα and mCherry/IIβ. In the negative controls, the presence of GFP but absence of mCherry/Iα and mCherry/IIβ indicates that the detection of mCherry-tagged proteins in the co-IP group was specific. The input analysis, which was used as a baseline to confirm the proper expression of all constructs in 293T cells, showed that the GFP antibody detected GFP/Ii (51.1 kDa) and GFP (26.3 kDa), and the mCherry antibody detected mCherry/Iα (62.8 kDa), mCherry/IIβ (51.8 kDa), and mCherry (26.0 kDa). Collectively, these results from the co-IP experiment and input analysis provide strong evidence for specific protein-protein interactions between GFP/Ii and both mCherry/Iα and mCherry/IIβ.
Identification of Ii-Iα/IIβ interactions via coimmunoprecipitation (co-IP) assay. Two sets of plasmids (pmCherry-N1-Iα and pEGFP-N1-Ii, pmCherry-N1-IIβ and pEGFP-N1-Ii, pmCherry-N1 and pEGFP-N1-Ii, pmCherry-N1-Iα and pEGFP-N1, and pmCherry-N1-IIβ and pEGFP-N1) were cotransfected into 293T cells. After 36 hours of incubation, proteins were harvested and divided into 2 groups: experimental co-IP group and control input group. For co-IP groups, lysates were incubated with anti–green fluorescent protein (GFP) antibody-conjugated protein A/G magnetic beads, followed by washing. Western blot was subsequently performed using anti-mCherry and anti-GFP antibodies for both groups. Co-IP: Anti-GFP detects GFP/Ii (51.1 kDa) and GFP (26.3 kDa); anti-mCherry identifies mCherry/Iα (62.8 kDa) and mCherry/IIβ (51.8 kDa) without free mCherry. Controls: GFP+/mCherry–. Inputs: All constructs expressed (GFP/Ii, GFP, mCherry/Iα, mCherry/IIβ, and mCherry).
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.12.0394
Localization and colocalization of duck MHC and Ii molecules in the organelles of cells
To localized duck MHC Iα, MHC IIβ, and Ii we performed confocal microscopy assays using 293T cells that were transfected to express GFP/endo or GFP-ER and mCherry-MHC Iα, mCherry-MHC IIβ, and mCherry-Ii. mCherry-tagged MHC Iα, MHC IIβ, or Ii colocalizing with GFP/ER or GFP/endo exhibits orange fluorescence due to red-green spectral overlap, whereas control mCherry/GFP-ER/endo pairs failing to colocalize retained distinct red or green signals, respectively (Figure 6). These observations demonstrate that MHC Iα, MHC IIβ, and Ii localize to both ER and endosomal compartments.
Localization of MHC Iα/MHC IIβ/Ii with organelle markers (A and B) and colocalization with Ii (C). A—Two sets of plasmids (pmCherry-N1-Iα/pEGFP-N1-Ii/pmCherry-N1-IIβ and pEGFP-N1–endoplasmic reticulum [ER]) were cotransfected into 293T cells. B—Two sets of plasmids (pmCherry-N1-Iα/pEGFP-N1-Ii/pmCherry-N1-IIβ and pEGFP-N1–endosome [endo]) were cotransfected into 293T cells. C—Two sets of plasmids (pmCherry-N1-Iα/pmCherry-N1-IIβ and pEGFP-N1-Ii) were cotransfected into 293T cells. After 36 hours, the cells were fixed and observed under an LMS880 confocal laser-scanning microscope (60X oil; scale bar: 50 μm). White arrowheads indicate colocalization.
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.12.0394
The fluorescence microscopy results in Figure 6 illustrate the subcellular localization patterns of relevant proteins in cells. In the experimental group, GFP/Ii with green fluorescence colocalized with mCherry/Iα and mCherry/IIβ (red fluorescent protein) exhibiting red fluorescence, resulting in orange fluorescence within the cells. In contrast, the control groups showed distinct fluorescence patterns, with GFP/Ii displaying green fluorescence and mCherry maintaining red fluorescence independently. These results support the above findings that MHC Iα and MHC IIβ interact with Ii in eukaryotic cells.
Discussion
Protein structural elucidation forms the mechanistic basis for functional interrogation. Through integrated sequence-structure analysis of duck MHC Iα, MHC IIβ, and Ii, this study biochemically validated their binding affinities and compartmentalized localization, whereas the biological ramifications of Ii-MHC Iα/IIβ complex formation require systematic exploration via loss-of-function and gain-of-function assays. The role of Ii in the assembly and maturation of MHC II molecules and the presentation of exogenous antigen peptides is well established.21,37,38 Emerging evidence indicates that the Ii in mice and chickens binds to MHC I molecules and facilitates crosspresentation of antigenic peptides through the MHC I pathway.25 Notably, recent investigations in teleost models revealed that fish Ii not only interacts with MHC class I complexes but also potentiates T-cell–mediated immunity via molecular coupling of T-cell–restricted antigenic peptides to the Ii protein, suggesting an evolutionarily conserved immunomodulatory mechanism across vertebrate lineages.36,39,40 The representative species selected in this study—ducks, chickens, pigs, and cattle—exhibit similar protein architectures. Notably, certain functional domains of MHC Iα, MHC IIβ, and Ii molecules demonstrate partial conservation across chickens, pigs, and cattle. These structural and functional parallels provide critical evolutionary insights for deciphering the biological roles of these immune-related molecules in ducks.15 The immunoregulatory mechanisms of Ii in MHC Iα/IIβ–mediated immunity primarily involve dual roles: facilitating antigen processing and presentation by MHC molecules and modulating their functional dynamics. Research results show that Ii possesses an intrinsic capacity to directly chaperone antigenic peptides for MHC binding, and this unique property underpins its pivotal role as a versatile carrier for antigenic peptide vaccine development.26,28,30 However, whether duck Ii exhibits analogous molecular chaperone activity remains an open question meriting systematic investigation.
In this study, 2 methods were used to verify the interactions between molecules. The prokaryotic expression proteins GST/Ii fusion protein demonstrate specific binding capacity to both His/MHC Iα and His/MHC IIβ, as validated by affinity pull-down assays. Major histocompatibility complex molecules have complex molecular structures, and the affinity of the MHC molecules to bind with the presented antigen peptide is regulated by conformation changes.41–43 Because prokaryotic expression products often exist in the inclusion body, there is no natural molecular structure. Since prokaryotic expression products frequently reside in inclusion bodies and thus lack their natural molecular structures, the pull-down assay presents certain limitations. To address this issue, we additionally incorporated the co-IP experiment. The co-IP results (Figure 5) revealed that Ii can bind to MHC Iα or MHC IIβ. This indicates that, regardless of whether they are prokaryotic or eukaryotic expression proteins, there exist binding relationships among them.
Colocalization experiments using organelle-targeting plasmids revealed that Ii, MHC Iα, and MHC IIβ mainly localize to the ER and endo (Figure 6). However, technical limitations of the organelle-specific tracer plasmids and antibodies restricted a more thorough mapping of their potential alternative subcellular locations. Compared to existing literature, our findings add new knowledge about protein localization, yet future studies could employ advanced techniques or more relevant cell models. We used laboratory-adapted human cell lines instead of native duck cells, which, while convenient for experimentation, might not reflect in vivo duck conditions accurately. Nevertheless, our results establish a basis for further exploring the relationship between Ii and MHC. Future work could focus on closing the gap between laboratory models and the natural biological setting to enhance our understanding of these proteins’ roles in immunity.
Duck Ii could bind duck MHC Iα and MHC IIβ, forming Ii-MHC Iα and Ii-MHC IIβ complexes, and the complexes can dissociate into single molecules after SDS treatment. Both MHC and Ii proteins exhibit direct interactions and can colocalize in the ER and endos.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org.
Acknowledgments
None reported.
Disclosures
The authors have nothing to disclose. No AI-assisted technologies were used in the composition of this manuscript.
Funding
This study was supported by a grant from the Key Research and Development Plan, Anhui Province (grant No. 202204c0602003), and the National Key Research and Development Program (grant No. 2023YFD1802200).
References
- 1.↑
Braciale TJ, Morrison LA, Sweetser MT, Sambrook J, Gething MJ, Braciale VL. Antigen presentation pathways to class I and class II MHC-restricted T lymphocytes. Immunol Rev. 1987;98:95–114. doi:10.1111/j.1600-065x.1987.tb00521.x
- 2.↑
Truong HV, Sgourakis NG. Dynamics of MHC-I molecules in the antigen processing and presentation pathway. Curr Opin Immunol. 2021;70:122–128. doi:10.1016/j.coi.2021.04.012
- 3.↑
Justesen S, Harndahl M, Lamberth K, Nielsen LL, Buus S. Functional recombinant MHC class II molecules and high-throughput peptide-binding assays. Immunome Res. 2009;5:2. doi:10.1186/1745-7580-5-2
- 4.↑
Johansen TE, McCullough K, Catipovic B, Su XM, Amzel M, Schneck JP. Peptide binding to MHC class I is determined by individual pockets in the binding groove. Scand J Immunol. 2010;46(2):137–146. doi:10.1046/j.1365-3083.1997.d01-102.x
- 5.↑
Biedrzycka A, Bielanski W, Cmiel A, et al. Blood parasites shape extreme major histocompatibility complex diversity in a migratory passerine. Mol Ecol. 2018;27(11):2594–2603. doi:10.1111/mec.14592
- 6.↑
Radwan J, Babik W, Kaufman J, Lenz TL, Winternitz J. Advances in the evolutionary understanding of MHC polymorphism. Trends Genet. 2020;36(4):298–311. doi:10.1016/j.tig.2020.01.008
- 7.↑
Granados DP, Sriranganadane D, Daouda T, et al. Impact of genomic polymorphisms on the repertoire of human MHC class I-associated peptides. Nat Commun. 2014;5:3600. doi:10.1038/ncomms4600
- 8.
Abelin JG, Keskin DB, Sarkizova S, et al. Mass spectrometry profiling of HLA-associated peptidomes in mono-allelic cells enables more accurate epitope prediction. Immunity. 2017;46(2):315–326. doi:10.1016/j.immuni.2017.02.007
- 9.↑
de Castro JAL. How ERAP1 and ERAP2 shape the peptidomes of disease-associated MHC-I proteins. Front Immunol. 2018;9:2463. doi:10.3389/fimmu.2018.02463
- 10.↑
O’Connor EA, Westerdahl H, Burri R, Edwards SV. Avian MHC evolution in the era of genomics: phase 1.0. Cells. 2019;8(10):1152. doi:10.3390/cells8101152
- 11.
Kaufman J. Unfinished business: evolution of the MHC and the adaptive immune system of jawed vertebrates. Annu Rev Immunol. 2018;36:383–409. doi:10.1146/annurev-immunol-051116-052450
- 12.↑
Miller MM, Taylor RL Jr. Brief review of the chicken major histocompatibility complex: the genes, their distribution on chromosome 16, and their contributions to disease resistance. Poult Sci. 2016;95(2):375–392. doi:10.3382/ps/pev379
- 13.↑
Westerdahl H, Waldenström J, Hansson B, Hasselquist D, von Schantz T, Bensch S. Associations between malaria and MHC genes in a migratory songbird. Proc Biol. 2005;272(1571):1511–1518. doi:10.1098/rspb.2005.3113
- 14.↑
Bonneaud C, Balenger SL, Russell AF, Zhang J, Hill GE, Edwards SV. Rapid evolution of disease resistance is accompanied by functional changes in gene expression in a wild bird. Proc Natl Acad Sci U S A. 2011;108(19):7866–7871. doi:10.1073/pnas.1018580108
- 15.↑
Abualrous ET, Sticht J, Freund C. Major histocompatibility complex (MHC) class I and class II proteins: impact of polymorphism on antigen presentation. Curr Opin Immunol. 2021;70:95–104. doi:10.1016/j.coi.2021.04.009
- 16.↑
Ren L, Yang Z, Wang T, et al. Characterization of the MHC class II alpha-chain gene in ducks. Immunogenetics. 2011;63(10):667–678. doi:10.1007/s00251-011-0545-5
- 17.↑
Yin LJ, Chen L, Luo Y, et al. Recombinant fiber-2 protein protects Muscovy ducks against duck adenovirus 3 (DAdV-3). Virology. 2019;526:99–104. doi:10.1016/j.virol.2018.10.011
- 18.↑
Qin S, Dunn PO, Yang Y, Liu H, He K. Polymorphism and varying selection within the MHC class I of four Anas species. Immunogenetics. 2021;73(5):395–404. doi:10.1007/s00251-021-01222-9
- 19.
Liu Z, Xie X, Li Z, Zhang L, Zhang N. Complex assembly, crystallization and preliminary X-ray crystallographic analysis of duck MHC class I complexed with a TUMV viral peptide. Res Vet Sci. 2020;132:312–317. doi:10.1016/j.rvsc.2020.07.009
- 20.↑
Wu Y, Wang J, Fan S, et al. Structural definition of duck major histocompatibility complex class I molecules that might explain efficient cytotoxic T lymphocyte immunity to influenza A virus. J Virol. 2017;91(14):e02511–e02516. doi:10.1128/JVI.02511-16
- 21.↑
Wang N, Waghray D, Caveney NA, Jude KM, Garcia KC. Structural insights into human MHC-II association with invariant chain. Proc Natl Acad Sci U S A. 2024;121(19):e2403031121. doi:10.1073/pnas.2403031121
- 22.↑
Sun Y, Yang B, Wen T, et al. ANXA10 sensitizes microsatellite instability-high colorectal cancer to anti-PD-1 immunotherapy via assembly of HLA-DR dimers by regulating CD74. Cell Biol Toxicol. 2025;41(1):25. doi:10.1007/s10565-024-09982-2
- 23.↑
Blander JM. Regulation of the cell biology of antigen cross-presentation. Annu Rev Immunol. 2018;36:717–753. doi:10.1146/annurev-immunol-041015-055523
- 24.
Schuren AB, Costa AI, Wiertz EJ. Recent advances in viral evasion of the MHC class I processing pathway. Curr Opin Immunol. 2016;40:43–50. doi:10.1016/j.coi.2016.02.007
- 25.↑
Basha G, Omilusik K, Chavez-Steenbock A, et al. A CD74-dependent MHC class I endolysosomal cross-presentation pathway. Nat Immunol. 2012;13(3):237–245. doi:10.1038/ni.2225
- 26.↑
Mensali N, Grenov A, Pati NB, et al. Antigen-delivery through invariant chain (CD74) boosts CD8 and CD4 T cell immunity. Oncoimmunology. 2019;8(3):1558663. doi:10.1080/2162402X.2018.1558663
- 27.
Lopez J, Anna F, Authié P, et al. A lentiviral vector encoding fusion of light invariant chain and mycobacterial antigens induces protective CD4+ T cell immunity. Cell Rep. 2022;40(4):111142. doi:10.1016/j.celrep.2022.111142
- 28.↑
Esposito I, Cicconi P, D’Alise AM, et al. MHC class II invariant chain-adjuvanted viral vectored vaccines enhances T cell responses in humans. Sci Transl Med. 2020;12(548):eaaz7715. doi:10.1126/scitranslmed.aaz7715
- 29.
Sorensen MR, Holst PJ, Pircher H, Christensen JP, Thomsen AR. Vaccination with an adenoviral vector encoding the tumor antigen directly linked to invariant chain induces potent CD4(+) T-cell-independent CD8(+) T-cell-mediated tumor control. Eur J Immunol. 2009;39(10):2725–2736. doi:10.1002/eji.200939543
- 30.↑
Nazerai L, Buus S, Stryhn A, Thomsen AR, Christensen JP. Efficient control of Zika virus infection induced by a non-replicating adenovector encoding Zika virus NS1/NS2 antigens fused to the MHC class II-associated invariant chain. Viruses. 2021;13(11):2215. doi:10.3390/v13112215
- 31.↑
Fougeroux C, Turner L, Bojesen AM, Lavstsen T, Holst PJ. Modified MHC class II–associated invariant chain induces increased antibody responses against Plasmodium falciparum antigens after adenoviral vaccination. J Immunol. 2019;202(8):2320–2331. doi:10.4049/jimmunol.1801210
- 32.↑
Ye L, Liu X, Rout SN, et al. The MHC class II-associated invariant chain interacts with the neonatal Fc gamma receptor and modulates its trafficking to endosomal/lysosomal compartments. J Immunol. 2008;181(4):2572–2585. doi:10.4049/jimmunol.181.4.2572
- 33.↑
Liu A, Pan Q, Li Y, et al. Identification of chicken CD74 as a novel cellular attachment receptor for infectious bursal disease virus in bursa B lymphocytes. J Virol. 2020;94(2):e01712–e01719. doi:10.1128/JVI.01712-19
- 34.
Tohme M, Maisonneuve L, Achour K, Dussiot M, Maschalidi S, Manoury B. TLR7 trafficking and signaling in B cells is regulated by the MHCII-associated invariant chain. J Cell Sci. 2020;133(5):jcs236711. doi:10.1242/jcs.236711
- 35.↑
David K, Friedlander G, Pellegrino B, et al. CD74 as a regulator of transcription in normal B cells. Cell Rep. 2022;41(5):111572. doi:10.1016/j.celrep.2022.111572
- 36.↑
Chen FF, Lin HB, Li JC, et al. Grass carp (Ctenopharyngodon idellus) invariant chain of the MHC class II chaperone protein associates with the class I molecule. Fish Shellfish Immunol. 2017;63:1–8. doi:10.1016/j.fsi.2017.01.030
- 37.↑
Bikoff EK, Huang LY, Episkopou V, van Meerwijk J, Germain RN, Robertson EJ. Defective major histocompatibility complex class II assembly, transport, peptide acquisition, and CD4+ T cell selection in mice lacking invariant chain expression. J Exp Med. 1993;177(6):1699–1712. doi:10.1084/jem.177.6.1699
- 38.↑
Anderson MS, Miller J. Invariant chain can function as a chaperone protein for class II major histocompatibility complex molecules. Proc Natl Acad Sci U S A. 1992;89(6):2282–2286. doi:10.1073/pnas.89.6.2282
- 39.↑
Jahn ML, Steffensen MA, Christensen JP, Thomsen AR. Analysis of adenovirus-induced immunity to infection with Listeria monocytogenes: fading protection coincides with declining CD8 T cell numbers and phenotypic changes. Vaccine. 2018;36(20):2825–2832. doi:10.1016/j.vaccine.2018.03.080
- 40.↑
Boilesen DR, Ragonnaud E, Laursen H, et al. CD8+ T cells induced by adenovirus-vectored vaccine are capable of preventing establishment of latent murine γ-herpesvirus 68 infection. Vaccine. 2019;37(22):2952–2959. doi:10.1016/j.vaccine.2019.04.034
- 41.↑
Sadegh-Nasseri S, Natarajan S, Chou CL, Hartman IZ, Narayan K, Kim A. Conformational heterogeneity of MHC class II induced upon binding to different peptides is a key regulator in antigen presentation and epitope selection. Immunol Res. 2010;47(1–3):56–64. doi:10.1007/s12026-009-8138-1
- 42.
Chen S, Li Y, Depontieu FR, et al. Structure-based design of altered MHC class II-restricted peptide ligands with heterogeneous immunogenicity. J Immunol. 2013;191(10):5097–5106. doi:10.4049/jimmunol.1300467
- 43.↑
Schulze MS, Anders AK, Sethi DK, Call MJ. Disruption of hydrogen bonds between major histocompatibility complex class II and the peptide N-terminus is not sufficient to form a human leukocyte antigen-DM receptive state of major histocompatibility complex class II. PLoS One. 2013;8(7):e69228. doi:10.1371/journal.pone.0069228
